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Abstract:

A method of manufacturing a microtitre plate with wells having
transparent bottoms, where the microtitre plate includes at least one
physical deformation between at least two adjacent wells.

Claims:

1. A method of manufacturing a microtitre plate, comprising the steps of:
using a mould and a plastic material, moulding a microtitre plate of the
plastic material with wells having transparent bottoms, wherein the
microtitre plate comprises a cross-talk reduction physical deformation
between at least two adjacent wells, the mould including a surface
physical deformation matching the cross-talk reduction physical
deformation of the microtitre plate, wherein the cross-talk reduction
physical deformation of the microtitre plate is formed by said molding
step.

2. The method of claim 1, wherein the microtitre plate is manufactured in
one piece by the moulding step.

3. The method of claim 2, wherein said moulding step is a one of the
group consisting of vacuuming moulding, injection moulding, and
compression moulding.

7. The method of claim 1, wherein the cross-talk reduction physical
deformation of the microtitre plate have a height or a depth between 0.1
times and 10 times of a thickness of the plastic material of the
microtitre plate.

8. The method of claim 1, wherein the cross-talk reduction physical
deformation of the microtitre plate has a height between 2 to 6 times of
the thickness of the plastic material of the microtitre plate.

9. The method of claim 7, wherein the cross-talk reduction physical
deformation of the microtitre plate has a ridge shape, the height of the
ridge shape being between 0.1 times and 10 times of the thickness of the
plastic material of the microtitre plate.

10. The method of claim 7, wherein the cross-talk reduction physical
deformation of the microtitre plate has a channel shape, the depth of the
ridge shape being between 0.1 times and 10 times of the thickness of the
plastic material of the microtitre plate.

11. The method of claim 1, wherein, in an analysis method measuring light
emitted in or transmitted through, the wells of the microtitre plate, the
cross-talk reduction physical deformation of the microtitre plate reduces
measured cross-talk by 50% as compared to an equal microtitre plate of
the plastic material with wells having transparent bottoms and free of
the cross-talk reduction physical deformation between at least two
adjacent wells.

12. The method of claim 1, wherein the cross-talk reduction physical
deformation of the microtitre plate is one of the group consisting of a
channel, a ridge, a hole, a slit and a step.

13. The method of claim 12, with plural of said physical deformation,
wherein the physical deformation surround the wells in a geometrical
pattern.

14. The method of claim 13, wherein the physical deformations surround
the wells in the form of circles, grids or squares.

15. The method of claim 12, wherein, in an analysis method measuring
light emitted in or transmitted through, the wells of the microtitre
plate, the cross-talk reduction physical deformation of the microtitre
plate reduces measured cross-talk by 50% as compared to an equal
microtitre plate of the plastic material with wells having transparent
bottoms and free of the cross-talk reduction physical deformation between
at least two adjacent wells.

16. The method of claim 12, wherein the analysis method utilizes one form
the group consisting of photometry, chemiluminescence, bioluminescence,
and fluorescence.

17. The method of claim 12, wherein the analysis method is
sequencing-by-synthesis.

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a division of copending application Ser. No.
12/918,825 filed on Aug. 23, 2010; which is the 35 U.S.C. 371 national
stage of International application PCT/SE2009/050198 filed on Feb. 23,
2009; which claims the benefit of U.S. provisional application Ser. No.
61/064,217 filed Feb. 22, 2008; which claims priority to EP application
08101878.0 filed on Feb. 22, 2008. The entire contents of each of the
above-identified applications are hereby incorporated by reference.

FIELD OF INVENTION

[0002] The present invention involves a microtitre plate with reduced
optical cross-talk between wells. The present invention also relates to
such microtitre plates for use in analysis methods using light emission
or transmission.

BACKGROUND

[0003] A large number of biotechnological analysis methods utilize
multi-well sample plates, commonly referred to as microtitre plates.
Separate reactions are performed in each well and the end products of the
reactions are used to interpret the result of the analysis. Alternatively
the reactions may be followed in real-time.

[0004] Such analytical methods may be performed in solution, i.e. all the
reactants are present in the solution. If the analyte to be detected is
present, the reactants give some sort of signal, such as appearance or
change in colour, light emission, turbidity or the like. One example is
Pyrosequencing® analysis, which involves detection of light emitted
by an enzyme cascade system after incorporation of a nucleotide into a
nucleic acid strand. The analytical methods may also be performed on the
solid phase of the well, e.g. in a so-called sandwich assay wherein a
molecule with affinity for an analyte is bound to the bottom of the well,
a sample possibly containing the analyte is added, a labelled molecule
with affinity for the analyte is added, excess label is washed away and
any remaining label is detected, possibly after addition of a developing
agent.

[0005] The signal to be detected in methods such as those above may
involve light emission from the well or light transmission through the
well. It is common to automatically detect such light below the bottom of
the well by means of various light detecting means. The microtitre plate
therefore has a transparent bottom to enable real-time light detection
from below whilst reagents can be dispensed from above. If the colour or
turbidity of the well's contents is to be measured, light may be emitted
from a light source above the plate.

[0006] The most convenient and economic method of manufacturing such a
plate is to produce it from a sheet of transparent material, for example
by vacuum-moulding or injection moulding. Thus the whole plate is
transparent. The transparent plastic can, however, guide light from one
well to another. This phenomenon is caused by internal reflection and
transmission of light and results in a false signal from a well where no
light signal should be produced. The result is an incorrect result. Such
reflection and transmission should therefore be avoided.

[0007] WO 94/21379 presents a construction where the walls of the wells,
inside and/or outside, are coated with a reflective material such as
metal, ceramic or a semiconductor. Alternatively, the plate is made from
a reflective material, or by placing a transparent microplate inside a
reflective plate. However, these constructions are expensive to produce.
Also, when using such a coating technique, there is a risk of coating the
bottom surface of the well. Additionally, a coating on the inside might
affect the desired biological/chemical process in the well. Said document
also states that the use of pigments to colour the plastic, thereby
reducing the transmittance or reflectance, is also known. These solutions
will increase the manufacturing cost if a transparent bottom is needed,
since the plate needs to be formed with two different materials.

[0008] U.S. Pat. No. 6,051,191 provides a microplate which reduces the
cross-talk through a construction containing two different polymers. The
well consists of a transparent polymer that is placed in a matrix of
opaque polymer. The opaque polymer covers the outside of the walls of the
wells, as well as the top surfaces between said wells, leaving a well
with a transparent bottom and opaque walls. While this microplate reduces
the cross-talk between the wells, it also requires a complex multistep
production process, which makes it expensive to produce.

[0009] All the above-mentioned microtitre plates could be used in
light-based analytical methods. However, there is a need for a microtitre
plate that is easy to manufacture, is cheap, and still reduces the
transmittance and/or reflectance of light when used in analytical methods
based on light detection or measurement.

SUMMARY OF THE INVENTION

[0010] It is an objective of the present invention to provide a microtitre
plate construction that facilitates real-time measurements and exhibits
reduced optical cross-talk. The manufacturing cost of the microtitre
plate should be low enough to make it disposable.

[0011] In a first aspect of the invention, a microtitre plate with wells
having transparent bottoms is provided, wherein said microtitre plate has
at least one physical deformation between at least two adjacent wells.
The physical deformations may have the shape of a channel, a ridge, a
hole, a slit or a step.

[0012] In a further embodiment, each surface between adjacent wells has at
least one physical deformation.

[0013] In a further embodiment, the physical deformations may surround the
wells in any geometrical pattern. This may be done, for example, in the
form of a grid or circles or squares.

[0014] The microtitre plate according to the present invention may
suitably be used for analysis methods measuring light emission or
transmission, such as chemiluminescence, bioluminescence, photometry
and/or fluorescence.

[0015] The present invention provides a microtitre plate with wells having
transparent bottoms that allow real-time measurements, including
simultaneous addition of reactants during the measurements. Cross-talk
reduction is accomplished via physical deformations between the wells.
The microtitre plate can be manufactured in one piece through
conventional processing techniques which means an easy manufacturing
process and low production costs.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 shows a microtitre plate, as known in the prior art, seen
from above (a) and a cross-section of said microtitre plate (b). The
dotted arrows indicate the distribution of wells.

[0017] FIG. 2. shows a cross-sectional view of a microtitre plate
according to the present invention provided with different physical
deformations and different combinations of the same.

[0018]FIG. 3 shows a cross-sectional view of a microtitre plate according
to the present invention provided with a construction including holes or
slits, and a combination of holes or slits with a ridge. Dotted lines
represent holes or slits.

[0019] FIG. 4 shows a microtitre plate according to the present invention
seen from above provided with a physical deformation surrounding a well.
The hatched areas represents the physical deformations.

[0020]FIG. 5 shows two microtitre plates according to Example 1. Plate 1
has a channel between the wells while Plate 2 does not.

[0021] FIG. 6 shows a microtitre plate according to Example 2.

[0022] FIG. 7 shows a microtitre plate according to the present invention
a) as seen from above, b) a cross-sectional view (Y-Y), c) a
cross-sectional view (X-X), and d) a perspective view.

DETAILED DESCRIPTION OF THE INVENTION

[0023] The present microtitre plate may have the general structure of a
conventional microtitre plate according to FIG. 1. A microtitre plate
would normally be a rectangular plastic multi-well plate, but other sizes
or shapes are also possible. To facilitate real-time measurements, the
well needs to have a transparent bottom in order to allow light to pass
through for detection. Detection from above is also possible. Since
emitted/transmitted light is spread in all directions, it is transmitted
and/or reflected via the plastic material into adjacent wells causing an
incorrect detection. This "cross-talk" should be reduced to obtain
accurate measurements.

[0024] Referring to FIG. 1, the top surface of the microtitre plate is
designated 1 and refers to the whole surface, while reference numbers 1a
or 1b indicate a surface between two adjacent wells 14. 1a is the upper
top surface while 1b is the lower top surface. These reference numbers
will be used when describing the present invention below. Such a
conventional structure for a microtitre plate is used as a basis for the
microtitre plate according to the present invention.

[0025] FIG. 2 shows an embodiment of the microtitre plate according to the
present invention comprising different physical deformations for top
surfaces 1a and 1b. One or more channels 12 or ridges 13 may be formed on
either surface. Channels may be formed on both surfaces at the same
point, thus reducing the thickness of the material. Also, the physical
deformations can be situated on only one side. Any combination of the
physical deformations shown in FIGS. 2a-e is possible.

[0026]FIG. 3 shows embodiments wherein one or more holes or slits 15 in
the top surface between the wells 14 are used to reduce the transmission
of light in the plate. The dotted lines represent the holes or slits.
Said holes or slits reduce the cross-talk between the wells 14, but
obviously do not surround the wells 14 entirely. FIG. 3 also shows a
combination of holes or slits 15 with a ridge 13.

[0027] FIGS. 4a-d show geometrical patterns of physical deformations
surrounding a well 14, the hatched areas 16 represent the physical
deformation. Both continuous and non-continuous patterns are possible.

[0028] The shape, height or depth of the physical deformation(s) and the
geometrical pattern in which they surround the well are not crucial, as
long as the deformations reduce the cross-talk. In the case the physical
deformations have the shape of a channel or a ridge, suitably the height
of the ridges, or the depth of the channels, is suitably between 0.1
times and 10 times of the thickness of the plastic material used for
manufacturing the microtitre plate, preferably between 0.5 to 10 times
and most preferably between 2 to 6 times the thickness of the plastic
material. Typically the plastic material is about 0.5 mm thick.

[0029] The physical deformations may be formed during the manufacturing
process of the microtitre plate by using a mould with the desired
structure. Preferably, the microtitre plate is manufactured in one piece
by vacuum moulding. Other suitable manufacturing techniques known to a
person skilled in the art may be used, such as injection moulding or
compression moulding.

[0030] The microtitre plate according to the present invention can
suitably be used in analysis methods depending on light detection,
measuring light emitted in, or transmitted through, the wells of the
microtitre plate, and at different wave lengths. Light can be detected
either from above or below the plate. Various light-emitting phenomena
can be analysed such as bioluminescence, chemiluminescence, photometry
and fluorescence. The present invention is also suitable for
sequencing-by-synthesis methods.

EXAMPLES

Ex. 1

[0031] A channel was formed between the wells. This channel stopped a
substantial proportion of any light guided through the plastic from
entering the neighbouring well.

[0032] Two plates were manufactured from the same plastic. Plate 1 had a
channel 12 between the wells whereas Plate 2 did not, as shown in FIG. 5.

[0033] Wells B6-8 were filled with 20 μl Annealing Buffer used to
perform a Pyrosequencing® reaction. Enzyme and substrate for
performing Pyrosequencing analysis were added to well B7. Pyrosphosphate
was dispensed into B7 in Dispensation 2, to give a high signal. Water was
dispensed into B6 and B8, which should not give a signal. In this way
only Dispensation 2 in well B7 should generate a signal. B6 and B8 were
used to measure cross-talk. The dispensations in the wells are described
in the table below.

TABLE-US-00001
Dispensation
Well 1 2 3
B6 Water Water Water
B7 Water Pyrophosphate Water
B8 Water Water Water

[0035] These results clearly show that the addition of channels between
wells (Plate 1) reduces the cross-talk in comparison with a plate without
channels between wells is used (Plate 2). The percent values given in
column on the right hand side represent the signal ratio between B6 or B8
and B7. When comparing the said ratios for the wells without channels and
with channels, the cross-talk was reduced by more than 50%.

Ex. 2

[0036] Slits 15 were made between wells as shown in FIG. 6.

[0037] All 9 wells were filled with 20 μl Annealing Buffer used to
perform a Pyrosequencing® reaction. Enzyme and substrate for
performing Pyrosequencing analysis were added to the central well. Three
dispensations were made into all the wells. All dispensations, except the
2nd dispensation into the central well, were water, which should not
give a signal. Pyrosphosphate was dispensed, as dispensation 2, into the
central well to give a high signal. The signals from all wells were
measured after each dispensation. The results are shown below.